Methods and composition for modulating flavonoid content

ABSTRACT

A method for manipulating the production of flavonoids in tomatoes by manipulating gene activity in the flavonoid biosynthetic pathway by expressing genes encoding chalcone isomerase, compositions for use in such a method and tomato plants having altered flavonoid levels are disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates generally to methods formanipulating the production of flavonoids in plants by manipulatingendogeneous and incorporated gene activity in the flavonoid biosyntheticpathway and compositions for use in such methods. In particular, itrelates to methods for increasing flavonoid levels by altering the levelof chalcone isomerase activity. Chalcone isomerase is an enzyme involvedin the biosynthetic pathway of flavonoids.

BACKGROUND OF THE INVENTION

[0002] Flavonoids form a large group of polyphenolic compounds, based ona common diphenylpropane skeleton, which occur naturally in plants.Included within this class of compounds are flavonols, flavones,flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols andstilbenes. The flavonoids are mostly present as glycosides.

[0003] In tomato fruits, the main flavonoid found is naringenin chalcone(Hunt et al, Phytochemistry, 19, (1980), 1415-1419). It is known toaccumulate almost exclusively in the peel and is simultaneously formedwith colouring of the fruit. In addition to naringenin chalcone,glycosides of quercetin and, to a lesser extent, kaempferol are alsofound in tomato peel.

[0004] Reports in the literature suggest that there is increasingevidence that flavonoids are potentially health-protecting components inthe human diet. Epidemiological studies suggest a direct relationshipbetween cardioprotection and increased consumption of flavonoids, inparticular flavonols of the quercetin and kaempferol type, from dietarysources such as onion, apples and tea (see, for example, Hertog et al,Lancet, 342 (1993), 1007-1011).

[0005] Flavonoids have been reported to exhibit a wide range ofbiological activities in vitro including anti-inflammatory,anti-allergic and vasodilatory activity (Cook et al, NutritionalBiochemistry, 7, (1996), 66-76). Such activity has been attributed inpart to their ability to act as antioxidants, capable of scavenging freeradicals and preventing free radical production. Within this group ofcompounds, those having the most potent antioxidant activity are theflavonols (Rice-Evans et al, Free Radical Research, 22, (1995),375-383). In addition, flavonoids can also inhibit the activity of keyprocesses such as lipid peroxidation, platelet aggregation and capillarypermeability (see Rice-Evans et al, Trends in Plant Science, 2, (1997),152-159).

[0006] Based on studies of this type, there is presently considerableinterest in the development of food products from plants rich in suchprotective flavonoids.

[0007] It would be desirable to produce plants which intrinsicallypossess elevated levels of health protecting compounds such asflavonoids in order to develop food products with enhanced protectiveproperties. Traditionally, the approach to improving plant varieties hasbeen based on conventional cross-breeding techniques, but these are slowas they require time for breeding and growing successive plantgenerations. More recently, recombinant DNA technology has been appliedto the general problem of modifying plant genomes to produce plants withdesired phenotypic traits. Whilst reference has been made in theliterature to the use of genetic manipulation techniques in modifyingthe flavonoid biosynthetic pathway, as discussed beneath, it is notablethat these attempts have been directed in general towards modifyingpigmentary anthocyanin production.

[0008] The flavonoid biosynthetic pathway is well established and hasbeen widely studied in a number of different plant species (see, forexample, Koes et al, BioEssays, 16, (1994), 123-132). Briefly, threemolecules of malonyl-CoA are condensed with one molecule ofCoumaroyl-CoA, catalysed by the enzyme chalcone synthase, to givenaringenin chalcone which rapidly isomerises, catalysed by chalconeisomerase, to naringenin. Subsequent hydroxylation of naringenincatalysed by flavanone 3-hydroxylase leads to dihydrokaempferol.Dihydrokaempferol itself can be hydroxylated to produce eitherdihydroquercetin or dihydromyricetin. All three dihydroflavonolssubsequently can be converted to anthocyanins (by the action ofdihydroflavonol reductase and flavonoid glucosyltransferase) oralternatively converted to flavonols such as kaempferol, quercetin andmyricetin by the action of flavonol synthase.

[0009] A schematic overview of the flavonoid biosynthetic pathway ispresented in appendix 1, FIG. 1.1.

[0010] The manipulation of flavonoid levels in plants by altering theexpression of a single flavonoid biosynthetic gene is disclosed byNapoli (1990, Plant Cell, 2:279-289). Napoli discloses the introductionof a chimeric chalcone synthase (CHS) gene into Petunia. Saidintroduction is described to result in a block in the anthocyaninbiosynthesis. The resulting transformed petunia plants thereforecontained lower levels of flavonoids than untransformed plants,presumably due to co-suppression of the endogeneous CHS activity.

[0011] Que (1997, Plant Cell, 9: 1357-1368) discloses a comparison ofthe effect of strong and weak promoters that drive sense chalconesynthase transgenes in large populations of independently transformedplants. It is shown that a strong transgene promoter is required forhigh frequency cosuppression of CHS genes and for the production of afull range of phenotypes.

[0012] Howles (1996, Plant Physiol. 112: 1617-1624) discloses the stablegenetic transfer of the flavonoid biosynthetic gene phenylalanineammonia-lyase (PAL) from french bean into tobacco. A proportion of theobtained transgenic tobacco plants is shown to display overexpression ofPAL activity. According to Howles PAL overexpressing plants do notcontain altered levels of flavonoids.

[0013] It has been disclosed by Tanaka et al (1395, Plant and CellPhysiology 36: 6, 1023-1031) that heterologous transformation ordihydroflavonol reductase (DFR) can be used for the production of plantswith altered levels of anthocyanins.

[0014] There is no disclosure in the literature of the manipulation offlavonoids in plants by means of overexpression of chalcone isomerase.

[0015] Accordingly, there remains a continuing need for the developmentof methods for enhancing the levels of flavonoids, in particularflavonols, in plants.

SUMMARY OF THE INVENTION

[0016] Therefore, in a first aspect, the invention provides a method forproducing a plant capable of exhibiting altered levels of flavonoidscomprising incorporating into said plant one or more gene sequencesencoding a protein with chalcone isomerase activity, or incorporating anucleotide sequence encoding a protein functionally equivalent thereto.

[0017] The invention also provides a plant having one or more transgeneseach encoding a protein with chalcone isomerase activity, or a proteinfunctionally equivalent thereto, incorporated into its genome such thatits ability to produce flavonoids is altered.

[0018] According to a highly preferred embodiment, the invention furtherprovides a tomato plant having one or more transgenes each encoding aprotein with chalcone isomerase activity, or a protein functionallyequivalent thereto, incorporated into its genome such that its abilityto produce flavonoids is altered.

[0019] Also provided is a transformed plant having enhanced flavonoidlevels, not being chalcones, particularly enhanced flavonol levelscompared to similar untransformed plants. Preferably the level of saidflavonoids, not being chalcones, in transformed plants is least 4 timeshigher than in similar untransformed plants, more preferred 5-100, mostpreferred 10-40 times higher than in similar untransformed plants.

[0020] Further provided is a fruit-bearing plant, particularly a tomatoplant, having flavonoids, particularly flavonols, in the peel of thefruit.

[0021] Seeds, fruits and progeny of such plants and hybrids are alsoincluded within the invention.

[0022] The invention further provides DNA constructs coding for aprotein with chalcone isomerase activity, or a functionally equivalentsequence of said DNA construct, operably linked to a promoter. Whentransformed into a plant cell, these constructs are useful foroverexpressing genes encoding proteins with chalcone isomerase activity,thereby altering the ability of the plant to produce flavonoids. Theinvention also provides for plants comprising these constructs togetherwith seeds, fruits and progeny thereof.

[0023] Food products such as sauces, dressings, ketchups and soups,comprising at least part of a plant prepared according to the inventionare also provided.

[0024] Also provided are skin and hair protective products comprising atleast part of a plant according to the invention.

[0025] Also provided are pharmaceuticals comprising at least part of aplant according to the invention.

[0026] Definition of Terms

[0027] As used herein, “plant” means a whole plant or part thereof, or aplant cell or group of plant cells. It will be appreciated that alsoextracts are comprised in the invention.

[0028] A “flavonoid” or a “flavonol” may suitably be an aglycon or aconjugate thereof, such as a glycoside, or a methyl, acyl, sulfatederivative.

[0029] A “protein with chalcone isomerase activity” is a protein beingcapable of enzymatically catalysing the conversion of a chalcone into aflavanone, for example narichalcone into naringenin.

[0030] A “gene” is a DNA sequence encoding a protein, including modifiedor synthetic DNA sequences or naturally occurring sequences encoding aprotein, and excluding the 5′ sequence which drives the initiation oftranscription.

[0031] A “DNA sequence functionally equivalent thereto” is any sequencewhich encodes a protein which has similar functional properties.

[0032] According to another embodiment, a functionally equivalent DNAsequence shows at least 50% similarity to the respective DNA sequence.More preferably a functionally equivalent DNA sequence shows at least60%, more preferred at least 75%, even more preferred at least 80%, evenmore preferred at least 90%, most preferred 95-100% similarity, to therespective DNA sequence.

[0033] According to the most preferred embodiment a functionallyequivalent DNA sequence shows not more than 5 base pairs difference tothe respective DNA sequence, more preferred less than 3, e.g. only 1 or2 base pairs different.

[0034] According to another embodiment a functionally equivalentsequence is preferably capable of hybridising under low stringentconditions to the respective sequence.

[0035] “Breaker” is the ripening stage corresponding to the appearanceof the first flush of colour on the green fruit.

[0036] “Operably linked to one or more promoters” means the gene, or DNAsequence, is positioned or connected to the promoter in such a way toensure its functioning. The promoter is any sequence sufficient to allowthe DNA to be transcribed. After the gene and promoter sequences arejoined, upon activation of the promoter, the gene will be expressed.

[0037] A “construct” is a polynucleotide comprising nucleic acidsequences not normally associated in nature.

[0038] An “altered” level of flavonoids is used throughout thisspecification to express that the level of specific flavonoids in thetransformed plants differs from the level of flavonoids present inuntransformed plants. Preferably the difference is between 0.1 and 100fold. It will be appreciated that the specific flavonoids as meant hereare flavonoids other than chalcones as said specific flavonoids areformed at the expense of chalcones.

[0039] Therefore in the specification where these flavonoids are meantreference will be made to “specific flavonoids”.

[0040] An “increased” level of flavonoids is used to indicate that thelevel of is preferably at least 4 times higher than in similaruntransformed plants, more preferred 5-100, most preferred 10-40 timeshigher than in similar untransformed plants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention may be more fully understood by referenceto the following description, when read together with the accompanyingdrawings in which:

[0042]FIG. 1 shows the levels of the two dominant flavonoids, rutin (A.)and narichalcone (B.) in FM6203 tomato peel during ripening. Resultsrepresent the means of three independent samples.

[0043]FIG. 2 shows the northern analysis of tomato fruit harvested atdifferent developmental stages, denoted as: green (G), breaker (B),turning (T) and red (R), and separated into peel and flesh. Leaves (L)were harvested from young tomato plants. RNA was isolated from thesamples, separated on formaldehyde-agarose gels, blotted and hybridisedwith petunia chs-a, chi and fls probes.

[0044]FIG. 3 shows the restriction maps of pFLAP10 and pFLAP50.

[0045]FIG. 4 shows the restriction maps of pBBC3, pBBC50 and pSJ89.

[0046]FIG. 5 shows the Southern blot of chromosomal DNA from tomato.Chromosomal DNA was isolated from young leaves of transgenic andnon-transgenic tomato plants. 10 μg DNA was digested with EcoRI,separated on an agarose gel and blotted onto a nylon filter. The DNA washybridised with a ³²P-labelled nptII specific probe andautoradiographed.

[0047]FIG. 6 shows typical HPLC chromatograms, recorded at 370 nm, ofhydrolysed extracts of (A.) peel and (B.) flesh tissue of plantstransformed with the control plasmid pSJ89. Peaks corresponding to thequercetin and kaempferol aglycons are indicated.

[0048]FIG. 7 shows a typical HPLC chromatogram, recorded at 360 nm, of anon-hydrolysed extract of peel tissue of a tomato plant transformed withthe control plasmid pSJ89. Peaks corresponding to rutin, quercetintrisaccharide and narichalcone are indicated.

[0049]FIG. 8 shows levels of quercetin in hydrolysed extracts of fleshof tomatoes transformed with either the control pSJ89 (G series oftransformed plants) or the pBBC50 (C series of transformed plants) geneconstructs.

[0050]FIG. 9 shows a typical HPLC chromatogram, recorded at 360 nm, of anon-hydrolysed extract of peel tissue of a tomato plant transformed withpBBC50 (plant number C87). The major peaks correspond to rutin (R),isoquercitrin (IQ), kaempferol rutinoside/quercetin glycoside (KR/QG)(co-eluting compounds) and a putative kaempferol glycoside (KG) aremarked.

[0051]FIG. 10 shows the proposed biosynthetic pathway for the productionof flavonoids.

[0052]FIG. 11 shows the graph of the data represented in table 2.

[0053]FIG. 12 shows restriction maps of plasmids pUCAP and pUCM2.

[0054]FIG. 13 shows the restriction map of plasmid pFLAP10.

[0055]FIG. 14. shows the multiple cloning site as altered in pUCM2 fromAscI to PacI in the 5′ to 3′ orientation.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention is based on the unexpected finding thatchalcone isomerase may be a rate limiting step in the production offlavonoids in tomato fruit.

[0057] We have surprisingly found that upon incorporation of a genesequence encoding for a protein with chalcone isomerase activity inplants, the subsequent overexpression of this protein leads to very high(sometimes even 50-100 fold) increase in the amount of flavonoids in thefruit of said plant.

[0058] Applicants have found that in ripening tomato fruit two dominantflavonoids can be detected: flavonol rutin and narichalcone, which bothaccumulate in the peel of tomato fruit. At no developmental stage weresignificant amounts of flavonoids detected in the flesh of fruit.Without wishing to be bound by any theory applicants believe that theaccumulation of narichalcone in the peel of fruit before decliningthrough the red and over ripe stages, is indicative that chalconeisomerase represents a rate limiting step in the formation offlavonoids.

[0059] A method for elucidation of the rate limiting step in flavonoidbiosynthesis is further illustrated in the examples.

[0060] Advantageously, by means of the invention, levels of specificflavonoids, more particularly flavonols, in plants, particularlytomatoes, may be altered. Preferably in the method according to theinvention the levels of flavonoids, more particularly flavonols, inplants, particularly tomatoes, are increased. Moreover, it has beenfound that the level of flavonoids, in particular the level of specificflavonols, may be increased specifically in the peel of tomato fruit,thereby producing tomatoes with enhanced nutritional, preservative andflavour characteristics.

[0061] Most preferred in the method according to the invention thetransformed plant exhibits increased levels of kaempferol and/orquercetin, or their glycosides or derivatives thereof.

[0062] It will be appreciated that the invention furthermore relates toa method for producing a plant capable of exhibiting altered levels offlavonoids, comprising incorporating into said plant a gene sequenceencoding for chalcone isomerase, thereby increasing the level offlavonoids by overexpression of said chalcone isomerase. Therefore itwill be understood that the invention encompasses said gene sequenceencoding for chalcone isomerase and any sequence functionally equivalentthereto. This group of sequences is in the course of this applicationalso referred to as “a gene comprising a nucleotide sequence encoding anenzyme with chalcone isomerase activity”.

[0063] Therefore according to a further embodiment the invention relatesto a method for producing a plant capable of exhibiting altered levelsof flavonoids comprising incorporating into said plant a gene comprisinga nucleotide sequence encoding an enzyme with chalcone isomeraseactivity.

[0064] According to a preferred embodiment said gene comprises anucleotide selected from:

[0065] (i) a nucleotide sequence, encoding an amino acid sequence havingat least 40% similarity, to seq ID No1;

[0066] (ii) a nucleotide sequence capable of hybridising under lowstringent conditions to a sequence selected from the group of sequencesset forth under (i) above;

[0067] (iii) a nucleotide sequence encoding a protein that isfunctionally equivalent to the protein encoded by seq ID no 1.

[0068] Seq, ID 1 is an amino acid sequence obtainable from PIR database,accession number SO4725, as published by van Tunen et al, EMBO J. 7,1257-63 1988.

[0069] More preferred said gene comprises the nucleotide sequenceencoding an amino acid sequence having at least 60% similaritypreferably at least 90%, more preferred at least 95% or even 98%,similarity to the sequence as set forth in seq ID No1 (amino acidsequence of chalcone isomerase).

[0070] According to a highly desired embodiment, the gene which isincorporated into the plant in the method according to the inventionencodes the amino acid sequence of chalcone isomerase from petunia asset forth in seq ID No 1.

[0071] According to a preferred embodiment said nucleotide sequencecomprises a sequence which has at least 50% similarity, more preferredat least 60%, even more preferred at least 75%, even more preferred atleast 80%, still more preferred at least 90%, most preferred at least95% or even 98-100% similarity to Sequence ID no 2, and whereby saidsequence encodes a protein having chalcone isomerase activity.

[0072] Although the percentage similarity referred to above assumes anoverall comparison between the sequence set forth in at least one of thesequences of Seq ID 1, Seq ID 2, it is clear that there may be specificregions within molecules being compared, having less than 60%similarity.

[0073] It will be appreciated that the invention extends to any plantwhich is amenable to transformation.

[0074] Therefore, according to another embodiment, the inventionrepaties to a plant having one or more transgenes, each encoding aprotein with chalcone isomerase activity or a protein functionallyequivalent thereto, incorporated into its genome such that its abilityto produce flavonoids is altered.

[0075] Preferably the plants according to the invention are suitable forhuman consumption. Suitable plants are for example vegetables, fruits,nuts, herbs, spices, infusion materials. Suitable vegetables are forexample from the Pisum family such as peas, family of Brassicae, such asgreen cabbage, Brussel sprouts, cauliflower, the family of Phaseolussuch as barlotti beans, green beans, kidney beans, the family ofSpinacea such as spinach, the family of Solanaceae such as potato andtomato, the family of Daucus, such as carrots, family of Capsicum suchas green and red pepper, and berries for example from the family ofRibesiaceae, Pomaceae, Rosaceae, for example strawberries, blackberries, raspberries, black current and edible grasses from the familyof Gramineae such as maize, and citrus fruit for example from the familyof Rutaceae such as lemon, orange, tangerine. Also preferred are plantswhich can form the basis of an infusion such as black tea leaves, greentea leaves, jasmin tea leaves. Also preferred is the tobacco plant.

[0076] A particularly preferred plant for use in the method according tothe invention is the tomato plant.

[0077] It will furthermore be appreciated that the sequence encoding aprotein with chalcone isomerase activity may be a genomic or cDNA clone,or a sequence which in proper reading frame encodes an amino acidsequence which is functionally equivalent to the amino acid sequence ofthe protein encoded by the genomic or cDNA clone. By “functionallyequivalent” is meant any DNA sequence which is capable of similarbiological activity. A functional derivative can be characterised by aninsertion, deletion or a substitution of one or more bases of the DNAsequence, prepared by known mutagenic techniques such as site-directedmutagenesis. The functionality can be evaluated by routine screeningassays, for example, by assaying the flavonoid content of the resultingtransgenic plant. An in vitro assay to determine chalcone isomeraseactivity has been described by van Weely (1983, Planta 159: 226-230).

[0078] Gene sequences encoding a gene sequence for proteins withchalcone isomerase activity for use according to the present inventionmay suitably be obtained from plants, in particular higher plants asthese generally possess a flavonoid biosynthetic pathway. Suitable genesequences can for example be obtained from petunia, maize, arabidopsis,alfalfa, pea, bean, grape, apple.

[0079] The gene sequences of interest are preferably operably linked(that is, positioned to ensure the functioning of) to one or moresuitable promoters which allow the DNA to be transcribed. Said promotersare preferably promoters useful to obtain overexpression of the proteinwith chalcone isomerase activity in said host plant. Suitable promoters,which may be homologous or heterologous to the gene (that is, notnaturally operably linked to the expressed gene encoding a chalconeisomerase protein or a functional equivalent thereof) useful forexpression in plants are well known in art, as described, for example,in Weising et al, (1988), Ann. Rev. Genetics, 22, 421-477). Promotersfor use according to the invention may be inducible, constitutive ortissue-specific or have various combinations of such characteristics.Useful promoters include, but are not limited to, constitutive promoterssuch as carnation etched ring virus (CERV), cauliflower mosaic virus(CaMV) ³⁵S promoter, or more particularly the enhanced cauliflowermosaic virus promoter, comprising two CaMV 35S promoters in tandem(referred to as “Double 35S”), or the GBSS (granular bound starchsynthase) promoter.

[0080] According to a preferred embodiment fruit specific promoters areused. Suitable fruit-specific promoters include the tomato E8 promoter(Deikman et al, (1988), EMBO J, 7, 3315-3320), 2A11 (Van Haaren et al,Plant Mol Biol, 21, 625-640), E4 (Cordes et al, (1989), Plant Cell, 1,1025-1034) and PG (Bird et al, (1988), Plant Mol. Biol., 11, 651-662)Nicholass et al, (1995), Plant Molecular Biology, 28, 423-435, pTOM96(ref), fpb11(WO-A-91/05054).

[0081] In another preferred embodiment, the promoter is a constitutiveenhanced 35S CaMV promoter.

[0082] It will be appreciated that accumulation of flavonoids may beinhibited by the rate of production of the amino acid phenylalanine, theprimary substrate in the synthesis of phenylpropanoids and subsequentflavonoids. In order to increase phenylalanine biosynthesis, genesencoding enzymes of the phenylalanine pathway that are insensitive tofeed-back regulation may be introduced as an optional additional step.

[0083] Preferably the desired gene sequences, operably linked torespective suitable promoters, are fused to appropriate expressionsequences to provide an expression cassette functional in a plant cellwhich can be introduced into a plant cell by any conventional planttransformation method.

[0084] Therefore the invention also relates to a DNA constructcomprising sequences encoding for a protein with chalcone isomeraseactivity, or a functionally equivalent sequence thereof, operably linkedto a promoter; and relates to plants, preferably tomato plantscomprising said DNA construct.

[0085] Accordingly, the invention provides in a further aspect anexpression cassette comprising as operably linked components in the5′-3′ direction of transcription at least one unit, comprising apromoter functional in a plant cell, a gene sequence encoding a proteinwith chalcone isomerase activity and a transcriptional terminationregulatory region functional in a plant cell.

[0086] The promoter and termination regulatory regions will befunctional in the host plant cell and may be heterologous (that is, notnaturally occurring) or homologous (derived from the plant host species)to the plant cell and the gene. Suitable promoters which may be used aredescribed above.

[0087] The termination regulatory region may be derived from the 3′region of the gene from which the promoter was obtained or from anothergene. Suitable termination regions which may be used are well known inthe art and include Agrobacterium tumefaciens nopaline synthaseterminator (Tnos), Agrobacterium tumefaciens mannopine synthaseterminator (Tmas) and the CaMN 35S terminator (T35S). Particularlypreferred termination regions for use according to the invention includethe tobacco ribulose bisphosphate carboxylase small subunit terminationregion (TrbcS) or the Tnos termination region.

[0088] Such gene constructs may suitably be screened for activity bytransformation into a host plant via Agrobacterium and screening forflavonoid levels.

[0089] Conveniently, the expression cassette according to the inventionmay be prepared by cloning the individual promoter/gene/terminator unitinto a suitable cloning vector. Suitable cloning vectors are well knownin the art, including such vectors as pUC (Norrander et al, (1983, Gene26, 101-106), pEMBL (Dente et al (1983), Nucleic Acids Research, 11,1645-1699), pBLUESCRIPT (available from Stratagene), pGEM (availablefrom Promega) and pBR322 (Bolivar et al, (1977), Gene, 2, 95-113).Particularly useful cloning vectors are those based on the pUC series.The cloning vector allows the DNA to be amplified or manipulated, forexample, by adding sequences. The cloning sites are preferably in theform of a polylinker, that is a sequence containing multiple adjacentrestriction sites, so as to allow flexibility in cloning.

[0090] In a particularly preferred embodiment, the individualpromoter/gene/terminator units are cloned into adjacent pairs ofrestriction sites in a suitable cloning vector.

[0091] Suitably, the nucleotide sequences for the genes may be extractedfrom any nucleotide database and searched for restriction enzymes thatdo not cut. These restriction sites may be added to the genes byconventional methods such as incorporating these sites in PCR primers orby sub-cloning.

[0092] Preferably the DNA construct according to the invention iscomprised within a vector, most suitably an expression vector adaptedfor expression in an appropriate host (plant) cell. It will beappreciated that any vector which is capable of producing a plantcomprising the introduced DNA sequence will be sufficient.

[0093] Suitable vectors are well known to those skilled in the art andare described in general technical references such as Pouwels et al,Cloning Vectors. A laboratory manual, Elsevier, Amsterdam (1986).Particularly suitable vectors include the Ti plasmid vectors.

[0094] Transformation techniques for introducing the DNA constructsaccording to the invention into host cells are well known in the art andinclude such methods as micro-injection, using polyethylene glycol,electroporation, or high velocity ballistic penetration. A preferredmethod for use according to the present invention relies onagrobacterium—mediated transformation.

[0095] After transformation of the plant cells or plant, those plantcells or plants into which the desired DNA has been incorporated may beselected by such methods as antibiotic resistance, herbicide resistance,tolerance to amino-acid analogues or using phenotypic markers.

[0096] Various assays may be used to determine whether the plant cellshows an increase in gene expression, for example, Northern blotting orquantitative reverse transcriptase PCR (RT-PCR). Whole transgenic plantsmay be regenerated from the transformed cell by conventional methods.Such transgenic plants having improved flavonoid levels may bepropagated and crossed to produce homozygous lines. Such plants produceseeds containing the genes for the introduced trait and can be grown toproduce plants that will produce the selected phenotype.

[0097] In accordance with a particular embodiment of the invention, thecloning vectors plasmid pUCM2 and pUCM3 were prepared by modifying thecloning vector pUCAP (Van Engelen et al, (1995), Transgenic Research, 4,288-290).

[0098] The invention furthermore relates to a plant having one or moretransgenes each encoding a protein with chalcone isomerase activity, ora sequence functionally equivalent thereto, incorporated into its genomesuch that its ability to produce flavonoids is altered.

[0099] The invention also encompasses a tomato plant prepared accordingto the method of the invention.

[0100] The following examples are provided by way of illustration only.

[0101] DNA manipulations were performed using standard procedures wellknown in the art, as described, for example, in Sambrook et al,Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbour Laboratory Press, 1989 (hereinafter “Sambrook”).

[0102] The following literature references are mentioned in theExamples:

[0103] Becker, D. et al. (1992) Plant Mol. Biol. 20: 1195-1197

[0104] Bovy, A. G. et al. (1995) Acta Hortic. 405: 179-189.

[0105] Fulton, T. M. et al. (1995) Plant Mol. Biol. Rep. 13: 225-227

[0106] Hanahan, D. (1983) J. Mol. Biol. 166: 557-580.

[0107] Hertog, M. G. L. et al. (1992) J. Agric. Food Chem. 40:1591-1598.

[0108] Hoekema, A. et al. (1985) Plant Mol. Biol. 5: 85-89

[0109] Jefferson, R. et al. (1987) Embo J. 6: 3901-3907

[0110] Loyd, A. et al (1992), Science 258, 1773-1775

[0111] Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15: 73-97

[0112] Sambrook, J. et al. (1989) Molecular Cloning. A laboratorymanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0113] Saul, M. W. et al. (1988) Plant Mol. Biol. Man. Al: 1-16 (Eds.

[0114] Gelvin S. B. and Schilperoort, R. A.) Kluwer Academic Pubs.,London Symmans et al (1990) Biotechnology 8, 217-221

[0115] Vancanneyt, G. et al (1990). Mol. Gen. Gen. 220, 245-250.

[0116] Van Engelen, F. et al. (1995) Transgenic R. 4: 288-290

[0117] VanTunen, A. J. et al. (1988) EMBO J. 7: 1257-1263

EXAMPLES Example 1 Plant Material

[0118] Plants of tomato line FM6203 and transformants are grown in soilin a glasshouse with a 16 hour photoperiod and a 23/18° C. day/nighttemperature.

Example 2 Bacterial Strains

[0119] The Escherichia coli strain used is:

[0120] DH5α supE44, (lac ZYA-ArgF)U169, 80lacZM15, hsdR17 (rk−, mk+),recA1, encA1, gyrA96, thi-1, relA1, deoR (Hanahan, 1983).

[0121] The Agrobacterium strain used is LBA4404 (Hoekema, 1985).

[0122] Transformation of E. Coli DH5α is performed using the method ofHanahan, (1983).

[0123] Transformation of Agrobacterium LBA4404 is performed using afreeze/thaw method according to Saul et al, (1988).

Example 3 Elucidation of the Rate-Limiting Step in Flavonol Productionin Tomato Fruit

[0124] The rate-limiting step in flavonol production in tomato fruit isdetermined using two complimentary approaches; high performance liquidchromatography (HPLC) analysis of flavonoids in ripening tomato fruitand northern analysis using probes for the flavonoid biosynthetic geneschalcone synthase (chs), chalcone isomerase (chi) and flavonol synthase(fls).

[0125] 3.1 Analysis of Flavonoids in Ripening Tomato Fruit by HPLC

[0126] 3.1.1 Harvest of Tomato Fruit

[0127] Tomato fruit are harvested at five stages of ripening (green,breaker (the ripening stage corresponding to the appearance of the firstflush of color on the green fruit), turning, red and over-ripe;corresponding to approximately 21, 28, 31, 46 and 55 days post anthesisrespectively). For discrimination between flavonoids in peel and fleshtissues, the outer layer of approximately 2 mm thick (i.e. cuticula,epidermal layer plus some sub-epidermal tissue) is separated from thefruit using a scalpel and classified as peel. The jelly and seeds arethen removed and the remainder of the fruit is classified as fleshtissue. After separation, tissues are quickly cut into pieces and frozenin liquid nitrogen before being ground into a fine powder using apre-cooled coffee grinder. Peel and flesh tissues are lyophilised for 24hr and then stored under desiccating conditions at 4° C. until use.

[0128] 3.1.2 Extraction of Flavonoids from Tomato Tissues

[0129] Determination of flavonoid glycosides and narichalcone(2′,4′,6′,4-tetrahydroxychalcone) in tomato fruit is carried out using anon-hydrolysing method as follows: 40 mg of freeze-dried tomato tissueis weighed and transferred to a 10 ml Pyrex glass tube. To each tube 4ml of 75% aqueous methanol acidified with HCl to pH 2 is added. Thetubes are closed with screw tops containing a Teflon inlay and incubatedat room temperature (20-25° C.) for 1 hr with continuous mixing on aroller band.

[0130] 3.1.3 High Performance Liquid Chromatography (HPLC) Conditionsfor Flavonoid Analysis

[0131] 1 ml of each tomato fruit extract is taken using a disposablesyringe and filtered through a 0.2 μm PTFE disposable filter (InacomInstruments BV, The Netherlands) before injection into the HPLC system.

[0132] The HPLC system consisted of a Waters 600E Multisolvent DeliverySystem (Waters Chromatography), a Promis autoinjector (SeparationsAnalytical Instruments BV) with a fixed 10 μl loop, and a Nova-Pak C₁₈(3.9×150 mm, particle size 4 μm) analytical column (WatersChromatography) protected by a Guard-Pak Nova-Pak C18 insert. Bothcolumns are placed in a LKB 2155 HPLC column oven (Pharmacia Biotech)set at 30° C. A photodiode array detector (Waters 996) is used to recordspectra of compounds eluting from the column on-line. The detector isset at recording absorbance spectra from 240 to 600 nm with a resolutionof 4.8 nm, at a time interval of 1 sec. Millennium 2010 ChromatographyManager (Waters Chromatography BV) is used to control the solventdelivery system and the photodiode array detector.

[0133] HPLC separation of flavonoids in non-hydrolysed extracts isperformed using a gradient of acetonitril in 0.1% TFA, at a flow rate of1 ml/min: 12.5-17.5% linear in 3 min, then 17.5-25% in 32 min and 25-50%in 2 min, followed by a 3 min washing with 50% acetonitril in 0.1% TFA.After washing, the eluent composition is brought to the initialcondition in 2 min, and the column is equilibrated for 6 min before thenext injection.

[0134] HPLC data are analysed using the software of the Millennium 2010Chromatography Manager. Absorbance spectra (corrected for baselinespectrum) and retention times of eluting peaks (with peak purity betterthan purity threshold value) are compared with those of commerciallyavailable flavonoid standards. Quercetin and kaempferol glycosides andnarichalcone are quantified based on absorption at 360 nm. Dose-responsecurves (0 to 20 μg/ml) were established to quantify these compounds inthe non-hydrolysed tomato extracts. Flavonoid levels in tomatoes arecalculated on a dry weight basis for peel and flesh tissues. With theHPLC system and software used, the lowest detection limit for flavonoidsin tomato extracts is about 0.1 μg/ml, corresponding with 10 mg/kg dryweight and 1 mg/kg fresh weight. Variation between replicate injectionsis generally less than 5%.

[0135] 3.1.4 Characterisation of the Flavonoid Content in RipeningTomato Fruit

[0136] Two dominant flavonoids are detected in the peel of ripe tomatofruit, the flavonol rutin (quercetin 3-rutinoside) and narichalcone,which are identified by their retention time (RT) and absorbancespectrum. At least four other flavonol glycosides are also identified inthe tomato peel extracts, albeit in much smaller quantities than rutinor narichalcone. A full identification of these minor flavonol glycosidespecies is described in Example 8.

[0137] In contrast, the flesh tissue from ripe tomato fruit containsonly traces of rutin, no other flavonoid species are detectable.

[0138] The levels of rutin and narichalcone in the peel during ripeningof tomato fruit are shown in FIG. 1. Rutin levels increase during tomatoripening reaching their highest levels in the over-ripe stage(approximately 1 mg/g dry weight of peel). Narichalcone is absent in thepeel of green fruit but increases sharply during coloring of the fruit,reaching levels of approximately 10 mg/g dry weight in peel of turningfruit before declining through the red and over-ripe stages. The enzymechalcone isomerase (CHI) is believed to be responsible for catalysingthe formation of naringenin from narichalcone in the flavonoidbiosynthetic pathway (FIG. 10). Applicants are of the opinion that theaccumulation of narichalcone suggests that in the peel of ripeningtomatoes CHI represents a rate limiting enzyme in the formation offlavonols.

[0139] 3.2 Northern Analysis of Ripening Tomato Fruit

[0140] Northern analysis is used to determine the endogenous expressionof the flavonoid biosynthetic genes chs, chi and fls during thedevelopment of FM6203 tomato fruit.

[0141] RNA is isolated from the peel and flesh of green, breaker,turning and red fruit and also young leaves according to the protocol ofvan Tunen (1988). For RNA gel blot analysis, 10 μg of RNA is loaded onformaldehyde agarose gels and electrophoresed overnight at 25V.Separated RNA is then blotted overnight onto Hybond N⁺ membrane(Amersham).

[0142]Petunia hybrida cDNA fragments encoding the following flavonoidbiosynthetic enzymes are used as probes: chalcone synthase (CHS-A),chalcone isomerase (CHI) and flavonol synthase (FLS). These fragmentsare obtained by RT-PCR on RNA extracted from closed flowers of Petuniahybrida W115 with primer combinations F15/F16 (chi-a), F13/F14 (chs-a)and F20/F21 (fls). The obtained PCR products are checked by sequenceanalysis.

[0143] Probes are labelled with ³²P and purified according to methodsgiven in Gibco Life Technologies RadPrime Labelling system. Blots arehybridised overnight at 55° C. and washed three times in 2×SSC, 0.1%SDS, 55° C., 30 min, before being exposed to X-ray film for 48 hr.

[0144] The results of the northern blot are shown in FIG. 2. Both thechs and fls transcripts are abundantly present in the peel of tomatofruit in all developmental stages tested. The level of these twotranscripts peaks during the breaker and turning stage of developmentand subsequently decreased in the red stage. The chi transcript level isvery low in the peel of all developmental stages. Without wishing to bebound by any theory, applicants believe that this is indicative that oneof the rate limiting steps in flavonoid biosynthesis in the peel may lieat the level of chi gene expression. This result is in agreement withthe observation that narichalcone (the substrate for CHI), accumulatedto high levels in breaker and turning stage fruit (Example 3.1).

[0145] In the flesh of tomato fruit, the levels of chs, chi and flstranscripts are very low, in agreement with the HPLC data which showedonly trace amounts of rutin in this tissue (Example 3.1).

[0146] Chs, chi and fls transcripts are present in low but detectablelevels in tomato leaves.

Example 4 Gene Constructs

[0147] 4.1 Strategy to Overexpress a Rate-Liming Step of FlavonolProduction in Tomato Fruit

[0148] During the early stages of ripening of tomato fruit narichalconeaccumulates in the peel of the fruit (Example 3.1). The enzymeresponsible for converting narichalcone into naringenin is CHI. Theexpression levels of the gene encoding CHI remain low throughoutripening of the fruit (Example 3.2) suggesting CHI may constitute arate-liming step in the production of flavonols in the peel of tomatofruit.

[0149] The strategy consists of increasing the production of flavonolsin tomato fruit by overexpression of the Petunia hybrida gene encodingCHI. The introduced gene is expressed under the control of theconstitutive enhanced CaMV 35S promoter (also called double P35s orPd35S).

[0150] 4.1 Cloning the chi Gene from Petunia Hybrida

[0151] The chi-a gene is amplified from plasmid pMIP41, which containsthe complete chi-a cDNA from Petunia hybrida inbred line V30 (Van Tunenet al. 1988), with primer combination F15/F16. These primers contain a5′ extension with a unique BamHI (F15) and SalI (F16) restriction site(Table 1). This results in a 0.73 kb chi-a fragment.

[0152] 4.2 Construction of the chi Gene Fusion

[0153] The Pd35S-chi-Tnos gene construct is made as follows. PlasmidpFLAP10, a pUC-derivative containing a fusion of the consitutiveenhanced CaMV 35S promoter (P35s), the maize cl gene (cl) and theAgrobacterium tumefaciens nos terminator (Tnos), is used as recipient ofthe chi-a gene (FIG. 3a). A description of the properties of plasmidpFLAP10 is given in FIG. 3. The amplified chi-a cDNA is digested withBamHI/SalI and the resulting 730 bp fragment is ligated in plasmidpFLAP10 digested with the same enzymes, thus replacing the cl gene withthe chi-a gene. The resulting plasmid is denoted pFLAP50 (FIG. 3b).

[0154] 4.2.1 Construction of pFLAP10

[0155] The C₁ gene fusion was cloned in plasmid pUCM2, a derivative ofplasmid pUCAP (Van Engelen et al. 1995, Transgenic Research 4, p.288-290), in which the multiple-cloning-site was altered (FIG. 12), inthree major steps. Said altered multiple cloning site is shown in FIG.14.

[0156] Firstly, Tnos was amplified by PCR from pBI121 with primers F12and AB13 (see Table 1). The resulting 250 bp product was cloned in pUCM2as a SalI/ClaI fragment. This resulted in plasmid pFLAP1.

[0157] Secondly, the cl gene was cloned as a BamHI/SalI fragmentupstream of Tnos in pFlap2 as follows. The cl gene was transferred as a2 kb EcoRI fragment from plasmid pAL77 (Loyd 1992) to high-copy plasmidpBluescript SK−, resulting in plasmid pBlC1. The cl gene was isolatedfrom pBlC1 as a 1.6 kb EcoRI/PacI fragment and adapters F7F8 and F9F10(Table 1) were ligated to each end of the fragment in order to addunique BamHI and SalI restriction sites on both ends of the gene and todestroy the EcoRI and PacI sites. The resulting BamHI/SalI cl fragmentwas cloned upstream of the nos terminator, resulting in plasmid pFLAP2.

[0158] Thirdly, Pd35s was cloned as a KpnI/BamHT fragment upstream of clin pFLAP2 as follows. To create a unique BamHI site at the 3′ end of thed35s promoter, plasmid pMOG18 (Symans et al 1990, Biotechnology 8, p.217-221) was digested with EcoRV/BamHI, thus removing the 3′ part of thed35s promoter and the gusA gene. The 3′ part of the 35S promoter presentin plasmid pAB80 (Bovy et al. (1995)) was ligated as a 0.2 kbEcoRV/BamHI fragment in the pMOG18 vector, resulting in plasmid pMOG18B.To Create a unique KpnT site at the 5′ end of the d35s promoter plasmidpMOG18B was digested with EcoRI, the ends were polished with Klenowpolymerase, and a subsequent digest with BamHI was done. The resulting0.85 kb blunt/BamHI d35s promoter fragment was cloned into plasmid pBlC1followed by digestion with XhoI and polished with Klenowpolymerase/BamHI. This resulted in plasmid pBld35S. Finally the d35spromoter was transferred as a KpnI/BamHI fragment from pBld35s toplasmid pFLAP2. This resulted in plasmid pFLAP10 (FIG. 13).

[0159] 4.3 Construction of Binary Vector pBBC3

[0160] To obtain a binary vector with suitable cloning sites to transferthe chi gene fusion into, plasmid pBBC3, a derivative of pGPTV-KAN(Becker et al. (1992)) is constructed as follows. Synthetic adapterF38F39 (Table 1) is ligated in plasmid pGPTV-KAN digested withEcoRI/HinDIII. In this way the gusA-Tnos gene in pGPTV-KAN is replacedby a small multiple-cloning-site consisting of PacI/EcoRI/HinDIII/AscIrestriction sites (FIG. 4a).

[0161] 4.4 Transfer of the chi Gene Fusion into pBBC3

[0162] The Pd35s-chi-Tnos insert is transferred from pFLAP50 as aPacI/AscI fragment into binary vector pBBC3, digested with the sameenzymes. The resulting binary plasmid is denoted pBBC50 (FIG. 4b).

[0163] 4.5 GPTV Control Plasmid

[0164] A GPTV-based binary plasmid (pSJ89) containing theβ-glucuronidase gene (with the st-ls1 intron; Vancanneyt et al. 1990)under control of the CaMV ³⁵S promoter and the nos poly(A) signal(P35s-gusA-Tnos) is used as a control plasmid to transform FM6203 (FIG.4c). This allows direct comparison between gus transformed controlplants and plants containing the chi construct as both sets of plantshave gone through the tissue culture procedure.

[0165] Plasmid pSJ89 is constructed as follows: the CaMV 35Spromoter—gus-int fragment (Vancanneyt et al, 1990) is cloned as aHindIII—SacI fragment into the same sites of plasmid pSJ34, a derivativeof the binary vector pGPTV-KAN (Becker et al, 1992) in which the BamHIsite between the NPTII selectable marker and the gene 7 poly(A) signalis destroyed by filling in with klenow polymerase.

Example 5 Stable Transformation of chi Construct into Tomato Line FM6203

[0166] 5.1 Agrobacterium Tumefaciens Transformations

[0167] Binary plasmids pBBC50 and pSJ89 are introduced intoAgrobacterium strain LBA4404 by adding 1 μg of plasmid DNA to 100 μl ofcompetent Agrobacterium cells, prepared by inoculating a 50 ml culturein YEP medium (Sambrook, 1989) and growing at 28° C. until the culturereaches an OD₆₀₀ of 0.5-1.0. The cells are then pelleted, resuspended in1 ml of CaCl₂ solution and dispensed into 100 μl aliquots. TheDNA-Agrobacterium mixture is frozen in liquid nitrogen and thawed in awater bath at 37° C. After the addition of 1 ml YEP medium the bacteriaare incubated at 28° C. for 4 hours with gentle shaking. Finallytransformed bacteria are selected on YEP-agar plates containing 50 μg/mlkanamycin. The presence of the plasmids is tested by PCR analysis usingpBBC50 (chi 5 and nos ant) or pSJ89 (300 35s and gus 2) specific primersrespectively (Table 1).

[0168] 5.2 Tomato Transformations

[0169] Seeds from tomato line FM6203 are sterilised by a 2 h incubationin 1.5% hypochlorite, followed by three rinses of sterile water. Theseeds are germinated and seedlings are grown for 8 days on a 1:1 mixtureof vermacolite and MS medium (Murashige and Skoog, 1962; Duchefa)supplemented with 0.3% (w/v) sucrose, with a photoperiod of 16 h (3000lux) at 25° C.

[0170] Eight-day old cotyledons are cut into 25 mm² squares andpreincubated for 24 h on tobacco suspension feeder layer plates at lowlight intensity (1000 lux). The tobacco leaf suspension culture is grownon plates containing MS medium including vitamins, supplemented withsucrose (3% w/v), agarose (6 g/l), 2,4-dichlorophenoxyacetic acid(2,4-D; 0.5 mg/l) and benzylaminopurine (BAP; 0.5 mg/l).

[0171] A single colony from the Agrobacterium LBA4404 culturescontaining one of the binary vectors mentioned in Example 4.4 and 4.5 isgrown for 48 h in liquid Minimal A medium (Sambrook, 1989) supplementedwith 50 μg/ml kanamycin to an OD₆₀₀ of 0.5-1.0. The bacteria arepelleted by centrifugation and resuspended in MS medium includingvitamins (Duchefa) and 3% (w/v) sucrose at an OD₆₀₀ of 0.5. Thecotyledon explants are incubated in the Agrobacterium suspension for 30min, blotted dry on filter paper and co-cultivated for 48 h on tobaccofeeder layer plates at 25° C. and low light intensity.

[0172] After co-cultivation, the explants are transferred toregeneration medium, consisting of MS medium supplemented with Nitschvitamins, sucrose (2% w/v), agargel (5 g/l), zeatin-riboside (2 mg/l),kanamycin (100 mg/l) and cefotaxime (500 mg/l). Regenerating explantsare transferred to fresh medium every two weeks. Regenerating kanamycinresistant shoots were transferred to rooting medium, consisting of MSmedium plus B5 vitamins, supplemented with sucrose (0.5% w/v), gelrite(2 g/l), kanamvcin (50 mg/l) and cefotaxime (250 mg/l). Duringregeneration and rooting explants are incubated in a growth chamber at25° C. with a 16 h photoperiod (3000 lux). After root formation, thepresence of the CHI insert is confirmed by PCR analysis of cotyledontissue using specific primers (chi 5 and nos ant), and the presence ofthe GUS insert using 300 35s and gus2 specific primers (Table 1). PCRpositive plantlets are transferred to soil and grown in the greenhouse.

[0173] Transgenic plants carrying the construct pBBC50 are numbered fromC6 onward. Control transgenic plants carrying the construct pSJ89 arenumbered from G2 onward.

Example 6 Southern Analysis of Transgenic Plants

[0174] The presence and the copy number of the transgenes is determinedin transgenic plants by southern hybridisation. Genomic DNA is isolatedfrom young leaves as described by Fulton et al., (1995). Aliquots of 10μg genomic DNA are digested for 16 h with EcoRI and separated on a 0.8%TAE agarose gel. The DNA is denatured in 0.5 M NaOH, 1.5M NaCl for 45min before being transferred to a Hybond N+ membrane (Amersham) in20×SSC.

[0175] The blots are probed with a 700 base pair ³²P radiolabelednptII-specific PCR fragment, amplified from plasmid pBBC3 with primersnpt IIa and npt IIb (Table 1), under stringent conditions (65° C.)Prehybridisation is carried out for 2 h at 65° C. in a mix of 0.5 MNa₂PO₄ pH 7.2, 7% SDS and 0.1 mg/ml denatured herring sperm DNA.Hybridisation is performed by adding denatured probe DNA to theprehybridisation medium and continuing the incubation at 65° C. for 16h. The hybridised blots are washed once for 30′ at 25° C. in 2×SSC, 0.1%SDS and then once for 30′ at 65° C. in 2×SSC, 0.1% SDS before beingautoradiographed.

[0176] The result of the southern analysis is shown in FIG. 5. Thecontrol is an untransformed FM6203 plant. Southern analysis confirms theinitial screening of transgenics by PCR (see Example 5.2), every pBBC50transformed plant hybridised with the npt II probe. Transgenic plantscontain either 1 or 2 copies of the insert.

Example 7 Measurement of Flavonoids in Transformed Tomato Plants

[0177] 7.1 Growth and Harvest of Tomato Fruits

[0178] Transgenic tomato plants are grown in 10 l cots in a glasshouseat standard growth conditions (day/night temperatures 23° C./18° C., 16hr light)). Fruits are harvested between 15-21 days post-breaker stage(corresponding to fully red ripe fruit. For discrimination betweenflavonoids in peel and flesh tissues, the outer layer of approximately 2mm thick (i.e. cuticula, epidermal layer plus some sub-epidermal tissue)is separated from the fruit using a scalpel and classified as peel. Thejelly and seeds are then removed and the remainder of the fruit wasclassified as flesh tissue. After separation, tissues are quickly cutinto pieces, frozen in liquid nitrogen and stored at −80° C. until use.

[0179] 7.2 Extraction of Flavonoids from Tomato Tissues

[0180] Flavonoids are determined as aglycons or as their glycosides bypreparing hydrolysed and non-hydrolysed extracts, respectively.

[0181] Acid hydrolysis is used as an initial screen of transformants inorder to identify those lines containing high amounts of flavonols ascompared to the control. Acid hydrolysis ensures that flavonoidglycosides such as rutin and kaempferol rutinoside are hydrolysed totheir respective aglycones i.e. quercetin and kaempferol.

[0182] Preparation of hydrolysed extracts is performed according toHertog et al (1992) with some modifications. Frozen tissues are groundinto a fine powder using a pre-cooled coffee grinder. Peel and fleshtissues are lyophilised for 24 h before flavonoid extraction. 50 mg ofthis freeze-dried material was weighed and transferred to a 6 ml Pyrexglass tube. To each tube 1.6 ml of 62.5% methanol (HPLC grade) indistilled water and 0.4 of 6 M HCl are added. The tubes are closed withscrew caps containing a Teflon inlay and incubated for 60 mm at 90° C.in a waterbath.

[0183] After hydrolysis, the tubes are cooled on ice, the extracts arediluted with 2 ml of 100% methanol and sonicated for 5 min. 1 ml of thesample was then filtered over a 0.2 μm PTFE disposable filter into astandard 1.8 ml HPLC vial.

[0184] Preparation of non-hydrolysed extracts is performed as follows:Frozen tissues are ground to a fine powder using a pre-cooled coffeegrinder. Peel and flesh tissues are lyophilised for 24 h beforeflavonoid extraction. 50 mg of freeze dried material is weighed andtransferred to a 6 ml Pyrex glass tube. 4 ml of 70% methanol (HPLCgrade) in distilled water is added to each tube. The tubes are closedwith screw top caps containing a Teflon inlay and placed in a sonicatingwater bath at room temperature for 30 min. After sonication 1 ml of thesample is filtered over a 0.2 μm PTFE disposable filter into a standard1.8 ml HPLC vial.

[0185] 7.3 HPLC Conditions for Flavonoid Analysis

[0186] Chromatography of samples is performed using a chromatographystation equipped with a dual pump system and automated gradientcontroller (model 1100; Hewlett Packard), a Waters auto-injector (model717) with a variable 20 μl loop and a Nova-Pak C₁₈ (3.9×150 mm, particlesize 4 μm) analytical column (Waters Chromatography) protected by aGuard-Pak Nova-Pak C18 insert. Both columns are placed in a LKB 2155HPLC column oven (Pharmacia Biotech) set at 30° C. A photodiode arraydetector (model 1040M, Hewlett Packard) is used to record spectra ofcompounds eluting from the column on-line. The detector is set atrecording absorbance spectra from 240 to 600 nm with a resolution of4.8=m, at a time interval of 1 second. Peak purity, identification andintegration were carried out on Hewlett Packard Chemstations softwareversion A.04.02.

[0187] HPLC separation of flavonoids present in hydrolyzed extracts(flavonols and naringenin) is carried out under isocratic conditions of25% acetonitril (for HPLC far UV) in 0.1% trifluoroacetic acid (TFA) ata flow rate of 0.9 ml/min.

[0188] HPLC separation of flavonoids in non-hydrolysed extracts(flavonoid-glycosides and narichalcone) is performed using a gradient ofacetonitril in 0.1% TFA, at a flow rate of 1.0 ml/min: 5-25% linear in30 min, then 25-30% in 5 min and 30-50% in 2 min followed by a 3 minwashing with 50% acetonitril in 0.1% TFA. After washing, the eluentcomposition is brought to the initial condition in 2 min, and the columnis equilibrated for 6 min before next injection.

[0189] HPLC data are analysed using the software of the Hewlett PackardChemstations software version A.04.02. Absorbance spectra (corrected forbaseline spectrum) and retention times of eluting peaks (with peakpurity better than purity threshold value) are compared with those ofcommercially available flavonoid standards. Quercetin and kaempferolaglycons are detected and calculated from their absorbance at 370 nm,naringenin at 280 nm and flavonol-glycosides as well as narichalcone at360 nm. Flavonoid levels in tomatoes are calculated on a dry weightbasis. With the HPLC system and software used, the lowest detectionlimit for flavonoids in tomato extracts is about 0.1 μg/ml,corresponding with 10 mg/kg dry weight and 1 mg/kg fresh weight.

[0190] Using flavonoid standards (obtained from Apin Chemicals Ltd,Abingdon, UK) it is established that during the hydrolysis step,aglycons are released from their respective glycosides for 100%, whilechemically converted into naringenin for more than 95%. Recoveries ofquercetin, kaempferol and naringenin standards added to peel or fleshextracts just before hydrolysis are more than 90%.

Example 8 Characterisation of the Flavonoid Content in Transgenic TomatoFruit

[0191] 8.1 Flavonoids in Peel and Flesh of Control Tomatoes

[0192] In hydrolysed extracts of control red fruit of variety FM6203transformed with pSJ89, both quercetin and kaempferol are present inpeel tissue (FIG. 6a). In contrast, the hydrolysed extracts of fleshtissue from this fruit contain only traces of quercetin with nodetectable levels of kaempferol (FIG. 6b). Without wishing to be boundby any theory, applicants believe that the small amount of quercetindetected in the hydrolysed extracts of flesh originates from thevascular tissue in the flesh. Chromatograms obtained at 280 nm (notshown) of the same extracts reveal a large peak of naringenin in thepeel, but not in the flesh. There is no significant difference in theidentity and quantity of flavonoids found in the controlpSJ89-transformed tomatoes and those found in untransformed tomatoes(data not shown).

[0193] In non-hydrolysed extracts of control tomatoes transformed withpSJ89, at least 5 different flavonol-glycosides as well as narichalconeare detected in the peel (FIG. 7). NMR-studies (not shown) prove thatthe peak at RT=16.9 min is rutin while the peak at 15.2 min is aquercetin-3-trisaccharide: rutin with apiose linked to the glucose ofthe rutinoside. The retention time and absorbance spectrum of the minorpeak at 17.3 min correspond with those of quercetin-3-glucoside, whilethose of the peak at 19.7 min correspond with kaempferol-3-rutinoside.The small peak at 20.4 min has an absorbance spectrum comparable tokaempferol-3-rutinoside, but its higher RT value indicates a yet unknownkaempferol-glycoside. The large peak at 33.2 min is narichalcone.Aglycons of quercetin and kaempferol, as well as naringenin (all presentin hydrolysed peel extracts) are not detectable in any of thenon-hydrolysed extracts. In the non-hydrolysed flesh sample only a smallpeak corresponding to rutin is detected (data not shown).

[0194] After comparing the flavonoid species in hydrolysed extracts withthose in non-hydrolysed extracts of the same tissue, we conclude thatthe presence of quercetin and kaempferol aglycons in the hydrolysedextracts results from hydrolysis of their respective glycosides; thepresence of naringenin in hydrolysed peel extracts results fromisomerization of narichalcone during the hydrolysis step (cf. Example7.3).

[0195] 8.2 Flavonoids in Fruits of Transformed Tomato Plants

[0196] To determine whether the pBBC50 construct was able to overcomethe suspected rate limiting step in flavonol production in tomato fruit,transformants are analysed for the presence of flavonoids in the fleshand peel of their fruits. This screening is performed by HPLC usinghydrolysed extracts. Thirty six independent plants transformed withpBBC50, as well as six control plants transformed with pSJ89 areanalysed.

[0197] Analysis of hydrolysed extracts of flesh samples from pBBC50transformed fruit reveals no significant increase in flavonoids comparedto the control pSJ89 fruit (FIG. 8). The differences in quercetinconcentration in tomato flesh that are shown in FIG. 8 are believed tobe within the experimental error. Said experimental error is believed tobe relatively high when working at low concentration near the detectionlimit of 20 μg/g DW flesh.

[0198] In contrast, analysis of the hydrolysed extracts of peel of thetomato fruit reveals that the presence of the pBBC50 construct resultsin a significant increase in the levels of both quercetin and kaempferoltype flavonols in a proportion of transformed plants (Table 2, FIG. 11).Hydrolysed extracts of pBBC50 transformed plants display a range of peelquercetin concentrations with one line expressing a 69 fold increaseover the pSJ89 transformed control lines (plant C20). The amount ofkaempferol present in the hydrolysed extracts of pBBC50 transformedplants correlates with their quercetin concentrations—lines with higherconcentrations of quercetin seem also to possess higher concentrationsof kaempferol in hydrolysed extracts of their peel. Applicants wish topoint out that the variety in concentrations of quercetin, kaempferoland naringenin as measured for the transformed plants is believed torepresent the common representation of a transgenic population.Applicants however wish to stress that the currently obtained dataclearly show an increase in the level of quercetin and kaempferol in thepeel of transformed plants.

[0199] The pBBC50 transformed plants also display a range of peelnaringenin concentrations (note that these measurements were carried outon hydrolysed extracts, therefore the naringenin was originally derivedfrom narichalcone as can be deduced from analysis of non-hydrolysedextracts cf. Example 8.1). In general, those transformants possessingincreased concentrations of flavonols in their peel also possessdecreased concentrations of naringenin when compared to the controlfruit (Table 2). That the decrease in naringenin concentrationscorrelates with an increase in flavonol concentrations in the peel ofthe pBBC50 transformed fruits strongly suggests that CHI no longerrepresents a rate-limiting step in these plants.

[0200] The significant increase in fruit flavonol levels seen in thepBBC50 transformed plants seems to reveal that, as suggested in Example2, CHI represents a major rate limiting step in the production offlavonols in tomato peel which has now been overcome by the heterologousexpression of the petunia chi gene.

[0201] Using non-hydrolysed extracts, subsequently it is analysed inwhich form the flavonoids accumulated in the tomato peel of pBBC50transformed plants. FIG. 9 shows an example of HPLC chromatogramsobtained with non-hydrolysed peel extracts from a pBBC50 transformedtomato. As with the control FM6203 peel, rutin (RT=16.0 min) representsthe major quercetin glycoside which accumulates in the peel of thepBBC50 transformed tomato. In addition, significant amounts ofisoquercitrin (quercetin 3-glucoside) (RT=17.4 min) also accumulate inthe pBBC50 peel. The peak at 19.7 min appeared to contain a mixture oftwo compounds: the retention time and absorbance spectra of the majorcomponent corresponded to that of kaempferol rutinoside, whilst that ofthe minor component has an absorbance spectrum comparable to that of aquercetin glycoside. The small peak at 20.3 min has an absorbancespectrum comparable to kaempferol-3-rutinoside, but its higher RT valueindicates a yet unknown kaempferol-glycoside.

[0202] Quercetin and kaempferol aglycons, all clearly present in thehydrolysed extracts, are not detectable in the non-hydrolysed peelextracts of pBBC50 transformed tomatoes. Therefore, applicants believethat these compounds are fully derived from hydrolysis of theirrespective glycosides. No anthocyanins accumulate in the transformed redtomatoes, as is obvious from the absence of any peak in thechromatograms recorded at 520 nm (not shown). TABLE 1 Overview of PCRprimers and adapters used. primer * sequence (5′ to 3′ ) gus2GCATCACGCAGTTCAACGCTG (SeQ ID 3) 300 35S CGCAAGACCCTTCCTCTATATAAG (SeQID 4) nos ant CCGGCAACAGGATTCAATCTT (SeQ ID 5) chi5 GGTCGTGCCATTGAGAAGTT(SeQ ID 6) nptII a GAGGCGATTCGGCTATGACTG (SeQ ID 7) npt IIbATCGGGAGCGGCGATACCGTA (SeQ ID 8) F7 AATTGCACCGGTCG (SeQ ID 9) F8GATCCGACCG (SeQ ID 10) F9 TAGCCATGGG (SeQ ID 11) F10 TCGACCCATGGCTAAT(SeQ ID 12) F12 CCCGTCGACTTTCCCCGATCGTTCAAACATTTGC (SeQ ID 13) F13CCCGGATCCAAAAATGGTGACAGTCGAGG (SeQ ID 14) F14CCGGTCGACGCAAATACATTCATGGCAAACG (SeQ ID 15) F15GGCGGATCCAAAAATGTCTCCTCCAGTGTC (SeQ ID 16) F16CCCGTCGACCTAAACTAGACTCCAATCACT (SeQ ID 17) F20CGGGGATCCAGAGGGCCTAACTTCTGTATAGAC (SeQ ID 18) F21CCCGTCGACTCGCGAAGATATAGCTAATCG (SeQ ID 19) F38AATTGGGCGCGCCAAGCTTCCGAATTCTTAATTAAG (SeQ ID 20) F39AGCTCTTAATTAAGAATTCGGAAGCTTGGCGCGCCC (SeQ ID 21) AB13CCCATCGATGCGTCTAGTAACATAGATGAC (SeQ ID 22)

[0203] TABLE II Flavonoid level in peel of transformed plants Line[Quercetin] μg/g [Kaempferol] μg/g [Naringenin] μg/g number dry weightpeel dry weight peel dry weight peel G4 115 15 280 G2 206 32 1095 G95210 45 980 G3 253 27 523 G96 265 45 625 G97 345 55 1150 C69 115 10 105C102 190 20 280 C41 208 28 789 C22 215 40 235 C54 230 40 410 C73 250 35930 C6 300 15 375 C10 301 37 383 C64 320 35 470 C24 325 35 545 C53 35065 345 C33 388 55 2107 C120 530 45 230 C51 690 217 43 C57 750 130 830C56 1490 100 270 C48 2530 215 60 C9 3155 95 80 C34 3375 175 75 C118 4355615 65 C25 4445 455 70 C88 4850 570 115 C86 4980 510 130 C39 5540 325 80C65 6203 596 80 C72 6372 951 20 C49 6970 735 50 C40 7244 1147 40 C677405 900 80 C38 7795 735 50 C11 7995 805 135 C103 8705 840 105 C10410055 870 110 C66 10885 1370 70 C87 13410 1250 95 C20 16520 2048 80

[0204]

1 23 1 241 PRT PETUNIA HYBRIDA 1 Met Ser Pro Pro Val Ser Val Thr Lys MetGln Val Glu Asn Tyr Ala 1 5 10 15 Phe Ala Pro Thr Val Asn Pro Ala GlySer Thr Asn Thr Leu Phe Leu 20 25 30 Ala Gly Ala Gly His Arg Gly Leu GluIle Glu Gly Lys Phe Val Lys 35 40 45 Phe Thr Ala Ile Gly Val Tyr Leu GluGlu Ser Ala Ile Pro Phe Leu 50 55 60 Ala Glu Lys Trp Lys Gly Lys Thr ProGln Glu Leu Thr Asp Ser Val 65 70 75 80 Glu Phe Phe Arg Asp Val Val ThrGly Pro Phe Glu Lys Phe Thr Arg 85 90 95 Val Thr Met Ile Leu Pro Leu ThrGly Lys Gln Tyr Ser Glu Lys Val 100 105 110 Ala Glu Asn Cys Val Ala HisTrp Lys Gly Ile Gly Thr Tyr Thr Asp 115 120 125 Asp Glu Gly Arg Ala IleGlu Lys Phe Leu Asp Val Phe Arg Ser Glu 130 135 140 Thr Phe Pro Pro GlyAla Ser Ile Met Phe Thr Gln Ser Pro Leu Gly 145 150 155 160 Leu Leu ThrIle Ser Phe Ala Lys Asp Asp Ser Val Thr Gly Thr Ala 165 170 175 Asn AlaVal Ile Glu Asn Lys Gln Leu Ser Glu Ala Val Leu Glu Ser 180 185 190 IleIle Gly Lys His Gly Val Ser Pro Ala Ala Lys Cys Ser Val Ala 195 200 205Glu Arg Val Ala Glu Leu Leu Lys Lys Ser Tyr Ala Glu Glu Ala Ser 210 215220 Val Phe Gly Lys Pro Glu Thr Glu Lys Ser Thr Ile Pro Val Ile Gly 225230 235 240 Val 2 729 DNA PETUNIA HYBRIDA 2 atgtctcctc cagtgtccgttactaaaatg caggttgaga attacgcttt cgcaccgacc 60 gtgaaccctg ctggttccaccaataccttg ttccttgctg gtgctgggca tagaggtctg 120 gagatagaag ggaagtttgttaagtttacg gcgataggtg tgtatctaga agagagtgct 180 attccttttc tggccgaaaaatggaaaggc aaaacccccc aggagttgac tgactcggtc 240 gagttcttta gggatgttgttacaggtcca tttgagaaat ttactcgagt tactatgatc 300 ttgcccttga cgggcaagcagtactcggag aaggtggcgg agaattgtgt tgcgcattgg 360 aaggggatag gaacgtatactgatgatgag ggtcgtgcca ttgagaagtt tctagatgtt 420 ttccggagtg aaacttttccacctggtgct tccatcatgt ttactcaatc acccctaggg 480 ttgttgacga ttagcttcgctaaagatgat tcagtaactg gcactgcgaa tgctgttata 540 gagaacaagc agttgtctgaagcagtgctg gaatcaataa ttgggaagca tggagtttct 600 cctgcggcaa agtgtagtgtcgctgaaaga gtagcggaac tgctcaaaaa gagctatgct 660 gaagaggcat ctgtttttggaaaaccggag accgagaaat ctactattcc agtgattgga 720 gtctagttt 729 3 21 DNAArtificial Sequence Description of Artificial SequencePRIMER 3gcatcacgca gttcaacgct g 21 4 24 DNA Artificial Sequence Description ofArtificial SequencePRIMER 4 cgcaagaccc ttcctctata taag 24 5 21 DNAArtificial Sequence Description of Artificial SequencePRIMER 5ccggcaacag gattcaatct t 21 6 20 DNA Artificial Sequence Description ofArtificial SequencePRIMER 6 ggtcgtgcca ttgagaagtt 20 7 21 DNA ArtificialSequence Description of Artificial SequencePRIMER 7 gaggcgattcggctatgact g 21 8 21 DNA Artificial Sequence Description of ArtificialSequencePRIMER 8 atcgggagcg gcgataccgt a 21 9 14 DNA Artificial SequenceDescription of Artificial SequenceADAPTER 9 aattgcaccg gtcg 14 10 10 DNAArtificial Sequence Description of Artificial SequenceADAPTER 10gatccgaccg 10 11 10 DNA Artificial Sequence Description of ArtificialSequenceADAPTER 11 tagccatggg 10 12 16 DNA Artificial SequenceDescription of Artificial SequenceADAPTER 12 tcgacccatg gctaat 16 13 35DNA Artificial Sequence Description of Artificial SequencePRIMER 13cccgtcgact ttccccgatc gttcaaacat ttggc 35 14 29 DNA Artificial SequenceDescription of Artificial SequencePRIMER 14 cccggatcca aaaatggtgacagtcgagg 29 15 31 DNA Artificial Sequence Description of ArtificialSequencePRIMER 15 ccggtcgacg caaatacatt catggcaaac g 31 16 30 DNAArtificial Sequence Description of Artificial SequencePRIMER 16ggcggatcca aaaatgtctc ctccagtgtc 30 17 30 DNA Artificial SequenceDescription of Artificial SequencePRIMER 17 cccgtcgacc taaactagactccaatcact 30 18 33 DNA Artificial Sequence Description of ArtificialSequencePRIMER 18 cggggatcca gagggcctaa cttctgtata gac 33 19 30 DNAArtificial Sequence Description of Artificial SequencePRIMER 19cccgtcgact cgcgaagata tagctaatcg 30 20 36 DNA Artificial SequenceDescription of Artificial SequenceADAPTER 20 aattgggcgc gccaagcttccgaattctta attaag 36 21 36 DNA Artificial Sequence Description ofArtificial SequenceADAPTER 21 agctcttaat taagaattcg gaagcttggc gcgccc 3622 30 DNA Artificial Sequence Description of Artificial SequencePRIMER22 cccatcgatg cgtctagtaa catagatgac 30 23 91 DNA Artificial SequenceDescription of Artificial SequenceMULTIPLE CLONING SITE 23 ggcgcgccaagcttgcatgc atcgatatgg tcgactctag aggatccccg ggtaccgagc 60 tcgaattccagatctgcggc cgcttaatta a 91

1. A method for producing a plant capable of exhibiting altered levelsof flavonoids comprising incorporating into said plant one or more genesequences encoding a protein with chalcone isomerase activity, orincorporating a nucleotide sequence encoding a protein functionallyequivalent thereto.
 2. Method according to claim 1 characterised in thatsaid gene or genes encode chalcone isomerase, isolated from a speciesselected from the group comprising tomato plant, petunia, maize,arabidopsis, alfalfa, pea, bean, grape, apple.
 3. Method according toclaim 1 characterised in that said gene or genes or nucleotide sequenceencode the protein chalcone isomerase, isolated from petunia.
 4. Amethod according to claim 1 characterised in that said plant is a tomatoplant.
 5. A method according to any one of claims 1 to 4 characterisedin that levels of “specific flavonoids” in said plant are increasedcompared to untransformed plants.
 6. A method according to any one ofclaims 1-5 characterised in that the level of “specific flavonoids” intransformed plants is at least 4 times higher than in similaruntransformed plants, more preferred 5-100, most preferred 10-40 timeshigher than in similar untransformed plants.
 7. A method according toany one of claims 1 to 6 characterised in that the level of specificflavonoids in the peel of the fruit of said plant is increased.
 8. Amethod according to any one of claims 1 to 7 wherein said flavonoid is aflavonol.
 9. A method according to any of claims 1 to 7 characterised inthat the flavonoid is quercetin or kaempferol or their glycosides or anyother derivative thereof.
 10. A method according to any one of claims 1to 8 characterised in that the introduced gene comprises a nucleotidesequence or complementary nucleotide sequence selected from: (i) anucleotide sequence, encoding an amino acid sequence having at least 40%similarity, to seq ID No1; (ii) a nucleotide sequence capable ofhybridising under low stringent conditions to a sequence selected fromthe group of sequences set forth under (i) above; (iii) a nucleotidesequence encoding a protein that is being functionally equivalent to theprotein encoded by seq ID no
 1. 11. A method according to claim 10,characterised in that said gene comprises a nucleotide sequence encodingan amino acid sequence having at least 60% similarity, preferably atleast 90%, more preferred at least 95%, most preferred at least 95%similarity to the sequence as set forth in seq ID No1.
 12. A methodaccording to claim 10, characterised in that said gene comprises anucleotide sequence, encoding an amino acid sequence having 99-100%similarity, to seq ID No1.
 13. A method according to any of claims 1-12,characterised in that said nucleotide sequence comprises a sequencewhich has at least 50, more preferably at least 60% similarity, morepreferred at least 75%, even more preferred at least 80%, still morepreferred at least 90%, most preferred 95-100% similarity to seq ID no2, and whereby said sequence encodes a protein having chalcone isomeraseactivity.
 14. A method according to any one of claims 1-13 characterisedin that the gene encoding a protein with chalcone isomerase is operablylinked to a promoter.
 15. A method according to claim 14 characterisedin that the promoter is selected from the group of: (a) constitutivepromoters, such as carnation edged ring virus, cauliflower mosaic virus35 S promoter, enhanced cauliflower mosaic virus 35 S promoter; (b)fruit specific promoters; (c) any other suitable promoter such as GBSS(granular bound starch synthase) promoter.
 16. A method according toclaim 15 characterised in that the promoter is a fruit specific promoterselected from the group of PG, 2A11, E8, E4, and fpb11.
 17. A methodaccording to any of claims 1-16 comprising the additional stepincreasing phenylalanine biosynthesis.
 18. A plant having one or moretransgenes, each encoding a protein with chalcone isomerase activity ora protein functionally equivalent thereto, incorporated into its genomesuch that its ability to produce flavonoids is altered.
 19. A plantaccording to claim 18, whereby said plant is a tomato plant, preparedaccording to the method of any one of claims 1 to
 17. 20. A DNAconstruct suitable for use to overexpress the encoded protein,comprising sequences coding for a protein with chalcone isomeraseactivity, or a functionally equivalent sequence thereof, operably linkedto a promoter.
 21. A plant comprising a DNA construct according to claim20.
 22. A transformed plant having enhanced levels of specificflavonoids compared to similar, untransformed plants.
 23. A tomato planthaving enhanced levels of specific flavonoids in the peel of the fruitcompared to the level of flavonoids in the peel of the fruit ofuntransformed plants.
 24. Seeds, fruits, progeny and hybrids of a plantaccording to any one of claims 18, 19, 21, 22, or
 23. 25. A food productcomprising at least part of a plant according to any one of claims 18,19, 21, 22, or
 23. 26. A food product according to claim 25characterised in that the food product is sauce, dressing, ketchup orsoup.
 27. A skin or hair protective product comprising at least part ofa plant according to any one of claims 18, 19, 21, 22, or
 23. 28. Apharmaceutical product comprising at least part of a plant according toany one of claims 18, 19, 21, 22, or 23.